Methods of treatment using ranolazine and related piperazine derivatives

ABSTRACT

Piperazine derivatives, particularly ranolazine, are useful for treatment of tissues experiencing a physical or chemical insult, and specifically for treating cardioplegia, hypoxic and/or reperfusion injury to cardiac or skeletal muscle or brain tissue, and for use in transplants.

This is a file wrapper continuation of pending application Ser. No.07/370,435, filed Jun. 23, 1989, incorporated herein by reference, nowabandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods of treatment using ranolazineor another piperazine derivative compound of Formula I, particularly tomethods of using ranolazine for treatment of tissues experiencing aphysical or chemical insult, and specifically to methods of treatingcardioplegia, hypoxic and/or reperfusion injury to cardiac or skeletalmuscle or brain tissue, and for use in transplants.

2. Background Information

Ranolazine, i.e.,±N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-1-piperazineacetamide or1-[3-(2-methoxyphenoxy)-2-hydroxypropyl]-4-[(2,6-dimethylphenyl)aminocarbonylmethyl]piperazine,and the dihydrochloride salt thereof, and the compounds of Formula I,are described in U.S. Pat. No. 4,567,264, incorporated herein byreference. Ranolazine is disclosed as a calcium entry blocking compounduseful in the treatment of cardiovascular diseases, such as, myocardialinfarction, congestive heart failure, angina and arrhythmia.

The anti-ischemic effects of ranolazine have been described in a numberof publications, such as, Jain et al., "A PRELIMINARY STUDY OF A NEWANTI-ANGINAL AGENT", Cardiovascular Drugs and Therapy, Vol. 1, No. 3, p.252 (October 1987); Allely and Alps, "THE EFFECTS OF THE NOVELANTI-ANGINAL AGENT ANOLAZINE (I.D.) IN A CANINE MODEL OF TRANSIENTMYOCARDIAL ISCHAEMIA", Br. J. Pharmacol., 1988, 93, 246P; and byFerrandon et al., PROTECTIVE EFFECTS OF THE NOVEL ANTI-ISCHAEMIC AGENTRANOLAZINE (RS-43285) IN PERFUSED RAT HEARTS", Br. J. Pharmacol. , 1988,93, 247P, where utility in protecting hearts from the potentially lethalbiochemical and functional injury produced by ischaemia and/orreperfusion was reported. Tissue protection, however, is not achieved bya calcium entry blockade nor by a beta-blockade mechanism (Brown et al.,Br. J. Pharmacol., 1988, 93, 248P), nor would such active agents beexpected to have a tissue protective effect. Moreover, cardiodepressionhas been identified as a limiting factor for extensive use of CEBs inthe treatment of cardio-related ischaemic conditions (Packer, et al.,Circn., 75(V), 56-64, 1987; Barjon, et al., J. Am. Coll. Cardiol.,622-630, 1987).

SUMMARY OF THE INVENTION

One aspect of the present invention concerns a method of treatingtissues experiencing a physical or chemical insult, by administering aneffective amount of a compound of Formula I: ##STR1## and thepharmaceutically acceptable esters and acid addition salts thereof,wherein:

R¹, R², R³, R⁴ and R⁵ are each independently hydrogen, lower alkyl,lower alkoxy, cyano, trifluoromethyl, halo, lower alkylthio, lower alkylsulfinyl, lower alkyl sulfonyl, N-optionally substituted alkylamido,except that when R¹ is methyl, R⁴ is not methyl; or

R² and R³ together form --OCH₂ O--;

R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each independently hydrogen, lower acyl,aminocarbonylmethyl, cyano, lower alkyl, lower alkoxy, trifluoromethyl,halo, lower alkylthio, lower alkyl sulfinyl, lower alkyl sulfonyl,di-lower alkyl amino;

R⁶ and R⁷ together form --CH═CH--CH═CH--; or

R⁷ and R⁸ together form --OCH₂ O--;

R¹¹ and R¹² are each independently hydrogen or lower alkyl; and

W is oxygen or sulfur,

In a preferred embodiment, the invention entails a method of treatmentwherein the compound of Formula I is one in which R¹ and R⁵ are methyl,particularly where R², R³, R⁴, R¹¹ and R¹² are hydrogen and moreparticularly where W is oxygen. Most preferred is the method oftreatment with ranolazine, i.e., where R⁶ is methoxy and R⁷, R⁸, R⁹ andR¹⁰ are hydrogen.

In another aspect, the present invention entails a method for protectingthe myocardium against global ischaemic damage induced by cardioplegia,which method comprises administering to a subject undergoing cardiacsurgery, and/or adding to the extra-corporeal circulation of suchsubject, an effective amount of a compound of Formula I, preferablyranolazine.

The invention also entails a method for protecting skeletal muscleagainst damage resulting, e.g., from trauma or subsequent to muscle orsystemic diseases, which method comprises administering to a subjectwhose skeletal muscle is or is likely to be damaged an effective amountof a compound of Formula I, preferably ranolazine.

A further aspect of this invention entails a method for treating shockconditions (including cardiogenic shock), which method comprisesadministering to a subject experiencing shock an effective amount of acompound of Formula I, preferably ranolazine.

Still another aspect of this invention entails a method of protectingmyocardial tissue against ischaemic damage in subjects with myocardialinfarction, especially in patients who are waiting to receive treatmentsuch as thrombolytic drugs or PTCA (percutaneous transluminal coronaryangioplasty), which method comprises administering to a subject at riskof myocardial tissue ischaemic damage an effective amount of a compoundof Formula I, preferably ranolazine.

Another aspect of the invention is a method for protecting neuronaltissue against ischaemia resulting from cardiac function impairment orfrom non-cardiac conditions (including protecting brain tissue againstischaemia-induced metabolic disorders), which method comprisesadministering to a subject suffering from or susceptible to suffer fromneuronal tissue damage an effective amount of a compound of Formula I,preferably ranolazine.

In still another aspect, the present invention entails a method forpreserving donor tissues used in transplants (protecting them from thedeleterious effects of ischaemia), by administration to the donor, therecipient and/or by adding to the ex-vivo perfusion fluid an effectiveamount of a compound of Formula I, preferably ranolazine or apharmaceutically acceptable salt thereof, particularly for renaltransplants, skin grafts, cardiac transplants, lung transplants, cornealtransplants, and liver transplants.

In yet another aspect, the invention relates to pharmaceuticalcompositions containing a therpeutically effective amount (up to 5 mg/mlfor liquid and semi-solid formulations) of a compound of Formula I,particularly ranolazine or a pharmaceutically acceptable salt thereof,admixed with at least one pharmaceutically acceptable excipient, suchcompositions being adapted for use in the methods of treatment of thepresent invention.

Another aspect of the invention entails methods of treatment bycoadministration of a compound of Formula I together with anotherpharmaceutically active agent, such as thrombolytic agents [especiallyTPA (Tissue Plasminogen Activator) or streptokinase] or anti-anginals(such as beta blockers, including propranolol and timolol).

DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Parameters

The following definitions are set forth to illustrate and define themeaning and scope of the various terms used to describe the inventionherein.

As used herein, the term "treatment" or "treating" means any treatmentof a disease in a mammal, including:

(i) preventing the disease, that is, causing the clinical symptoms ofthe disease not to develop;

(ii) inhibiting the disease, that is, arresting the development ofclinical symptoms; and/or

(iii) relieving the disease, that is, causing the regression of clinicalsymptoms.

As used herein, the term "q.s." means adding a quantity sufficient toachieve a stated function, e.g., to bring a solution to the desiredvolume (i.e., 100%).

As used herein, the term "effective amount" means a dosage sufficient toprovide treatment for the disease state being treated. This will varydepending on the patient, the disease and the treatment being effected.

Preparation of Ranolazine

Ranolazine and the piperazine compounds of Formula I can be prepared,for example, as described in U.S. Pat. No. 4,567,264, previouslyincorporated herein by reference.

Utility, Testing and Administration

It has surprisingly been found that ranolazine is active in methods oftreatment unrelated to its initially identified calcium entry blockingmechanism and cardioselective indications. Particularly interesting isthe fact that ranolazine has now been found to protect tissues againstischaemia (improving cellular oxygen utilization efficiency) at dosesthat do not produce any cardiodepressant effects (see, Allely and Alps,supra. and Ferrandon, et al., supra.)

General Utility

The piperazine compounds of Formula I, particularly ranolazine and thepharmaceutically acceptable salts thereof (preferably thedihydrochloride), are useful for treating tissues experiencing aphysical or chemical insult. For example, such treatment can be forcardioplegia, or for hypoxic reperfusion injury to cardiac or skeletalmuscles, or brain tissue. The compounds of Formula I, particularlyranolazine and its salts, are also useful for preserving (e.g.,preventing deterioration of) donor tissues used in transplants, byadministration to the transplant donor, to the transplant recipient, orby perfusion of the tissues to be transplanted, particularly for renaltransplants, skin grafts, cardiac transplants, lung transplants, cornealtransplants, and liver transplants.

Testing

Protection of the myocardium against ischaemic damage is experimentallydemonstrated by inducing infarction in a suitable test animal (e.g., ababoon) followed by examination of insult-induced elevations in enzymelevels (particularly creatine kinase "CK" and lactate dehydrogenase"LDH"). It is accepted that concentrations of these enzymes areincreased after myocardial damage (Galen, et al., J.A.M.A., 232,145-147, 1975) and that such enzyme levels can be measured by anexperimental test that is an adaptation of the one described by Alps, etal., (Arzneim. Forsch Drug Res., 33, (1), 6, 868-876, 1983). Thecompounds of Formula I, as exemplified by ranolazine, are active inreducing CK and LDH enzyme levels as measured by this assay.

Protection against myocardial ischaemia can also be assessed viaeffectiveness to prevent ischaemia-induced increase in alpha-1adrenoceptor number in the myocardium. It is known that alpha-1adrenoceptor population increases in the myocardium suffering fromischaemia (Heathers, et al., Circulation Research, 61, 735-746, 1987).It has also been shown that alpha-1 adrenoceptor antagonists havebeneficial effects during ischaemia in animal models (Wilbur, et al., J.Cardiovascular Pharmacol., 10, 96-106, 1987). Thus agents which preventthe ischaemia-induced increase in alpha-1 adrenoceptors density arebeneficial during myocardial ischaemia. The ability of compounds ofFormula 1, as exemplified by ranolazine, to inhibit theischaemia-induced increase of alpha-1 adrenoceptors in myocardium isassessed in the rat left ventricle using a model of ischaemia describedby Allely and Brown (Br. J. Pharmacol., 95, 705P, 1988). A detaileddescription is set forth in Example 6.

Protection of skeletal muscles against damage resulting, for example,from major surgical practices, was experimentally assessed in the samemodel used to assess its protective effects at the myocardial level. Forthis purpose skeletal muscle-specific isoenzymes CK₃ and LDH₅, areassayed as indications of damaged muscle (Galen, Med. Times, 105(2),89-99, 1977). A detailed description is set forth in Example 2.

Protection of the myocardium against deleterious effects of ischaemiainduced by open-heart and other cardiac surgical procedures, includingcardioplegia, is assessed by a method modified from Langendorff, whichentails measuring coronary effluent pH and lactate level. These tracersare recognised as indicative of tissue damage induced by severereduction in the nutrient supply to the heart (Armiger, et al., Biochem.Med., 29, 265-267, 1983; van Gilst, et al., Archives of Pharmacol.,suppl., 330, 161P, 1985). A detailed description is set forth in Example3.

The utility of compounds of Formula I, as exemplified by ranolazine, inorgan transplant is demonstrated by administering the test compound topigs before nephrectomy, and/or by adding the compound to the fluid usedfor flushing and storage of the organ and by assessing functionality oftransplanted kidneys over a period of 14 days. Improvement of renalfunction in treated animals is assessed by measurement of the glomerularfiltration rate and also by peak serum levels for creatinine and urea.Glomerular filtration is a well established indicator of renal function(see, e.g., Mudge and Weiner in The Pharmacological Basis ofTherapeutics, Goodman and Gilman, 879, 7th Ed, 1985) and it is generallyassessed by measurement of inulin and/or creatinine clearance (Textbookof Medicine, 1088-93, 14th Ed., 1975--Beeson and McDermott Editors). Adetailed description is set forth in Example 4.

Cerebral ischaemia is the result of either a generalized or a localizedprolonged reduction of blood flow to the brain. Such a blood flowreduction can result from various pathological conditions includingcerebral venous inflammation and thrombosis, cardiac diseases, changesin blood (clotting, viscosity, anaemia) or from cardiac surgicalpractices. One of the indications of damage produced by cerebralischaemia is the increase of the iso-enzyme creatinephosphokinase 1(CPK₁) in the plasma (Rossi, et al., Am. J. Cardiol., 58(13), 1236-1241,1986). Inhibition of the peripheral appearance of CPK₁ is an indicationof reduced damage caused by ischaemia to the brain. This is demonstratedby administration of a test compound prior to coronary artery ligationin the baboon, as a bolus i.v. injection followed by an infusion overthe period of reperfusion, as described by Alps, et al., (Arzneim.Forsch Drug Res., 33, (1), 6, 868-876, 1983).

Administration

Administration of ranolazine in pure form or in an appropriatepharmaceutical composition can be carried out via any of the acceptedmodes of administration of agents for serving similar utilities. Thus,administration can be, for example, orally, nasally, parenterally ortopically, including by perfusion. Administration can be achieved in theform of solid, semi-solid, lyophilized powder, or liquid dosage forms,such as for example, tablets, suppositories, capsules, powders,solutions, suspensions, emulsions, creams, lotions, aerosols, ointmentsor the like, preferably in unit dosage forms suitable for simpleadministration of precise dosages. The compositions will include aconventional pharmaceutical carrier or excipient and an effective amountof ranolazine or a pharmaceutically acceptable salt thereof, and inaddition, may include other medicinal agents, pharmaceutical agents,carriers, adjuvants, etc. Sustained release and slow releaseformulations for maintaining extended or constant dosage levels are alsouseful in the present invention. Ranolazine can also be co-administeredother active agents, such as thrombolytic agents [especially TPA (TissuePlasminogen Activator) or streptokinase] or anti-anginals (such as betablockers, including propranolol and timolol).

The preferred method of administration is parenteral, except for thosecases when the subject must be pre-treated before surgery or when thesubject must be maintained under therapy after acute episodes ofischaemia (in which instances it may be preferable to administer thecomposition orally).

Generally, depending on the intended mode of administration, thepharmaceutically acceptable compositions will contain about 1% to about99% by weight of the pharmaceutically active compound of this inventionand 99% to 1% by weight of suitable pharmaceutical excipients.Preferably, the composition will be about 5 to 75% by weight of apharmaceutically active compound, with the rest being suitablepharmaceutical excipients. For liquid and semi-solid formulations, about5 mg/ml is preferred as the maximum concentration for the activeingredient.

Oral administration entails using a convenient daily dosage regimenwhich can be adjusted according to the degree of affliction. For suchoral administration, a pharmaceutically acceptable, non-toxiccomposition is formed by the incorporation of any of the normallyemployed excipients, such as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, andthe like. Such compositions take the form of solutions, suspensions,tablets, capsules, powders, sustained release or slow releaseformulations and the like.

Preferably the oral compositions will take the form of a capsule ortablet and thus the composition will contain, along with the activeingredient, a diluent such as lactose, sucrose, dicalcium phosphate, andthe like; a disintegrant such as starch or derivatives thereof; alubricant such as magnesium stearate and the like; and a binder such asa starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose andderivatives thereof, and the like.

The active compounds may be formulated into a suppository using, forexample, about 0.5% to about 50% active ingredient disposed in a carrierof polyethylene glycols (PEG) [e.g., PEG 1000 (96%) and PEG 4000 (4%)]or semi-synthetic glycerides (Witepsol, suppocire).

Another preferred mode of administration is parenterally. Liquidpharmaceutically administerable compositions can, for example, beprepared by dissolving, dispersing, etc. an active compound (about 0.5%to about 20%), as described above, and optional pharmaceutical adjuvantsin a carrier, such as, for example, water, saline, aqueous dextrose,glycerol, ethanol and the like, to thereby form a solution orsuspension.

For preservation of tissues awaiting transplantation, a perfusionsolution is preferred. Such solutions include an active compound in acarrier such as Eurocollins Solution (Fresenius, A.G., Bad Homburg, vdH,Germany), University of Wisconsin Fluid (Kalayoglu, M., et al., TheLancet, 1988 i, 617), phosphate buffered sucrose (see, e.g., Example 7E)and Hyperosmolar Citrate (Ross, et al., Transplantation, 1976, 498-501).

If desired, the pharmaceutical composition to be administered may alsocontain minor amounts of non-toxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents and the like, such as forexample, sodium acetate, sorbitan monolaurate, triethanolamine oleate,etc.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences., 16th Ed., (Mack Publishing Company, Easton,Pa., 1980). The composition to be administered will, in any event,contain a quantity of the active compound(s) in a pharmaceuticallyeffective amount for relief of the particular condition being treatedwhen administered in accordance with the teachings of this invention.

Example 7 describes oral and parenteral formulations containingranolazine. Such formulations should not be construed as narrowing theinvention. In particular, parenteral formulations can be given asdilutions with perfusion fluids, dyalisis fluids and/or fluids used toflush and store organs. It is also intended that the inventionencompasses the possibility to associate ranolazine with otherpharmaceutical agents, as co-prescription or by concomitant dissolutionin fluids.

Dosage

Generally, ranolazine is administered in a therapeutically effectiveamount, i.e., a dosage sufficient to effect treatment. The amount ofactive compound administered will, of course, be dependent on thesubject treated, the subject's weight, the severity of the affliction,the route of administration and the judgement of the prescribingphysician. However, absent sufficient time to weigh the foregoingfactors in detail, e.g., in emergency situations, effective i.v. dosagesrange from about 0.05 to about 5 mg/kg for bolus injection followed byan infusion ranging from about 0.3 to about 30 mg/kg/hour. Preferably,the i.v. bolus dosage ranges from about 0.1 to about 2.5 mg/kg and theinfusion dosage from about 1.5 to about 15 mg/kg/hour. For an average 70kg human, the i.v. bolus would range from about 3.5 to about 350 mg, orpreferably, from about 15 to about 105 mg. In other situations, the oraldosage is in the range of about 35 to about 1400 mg per day, preferablyabout 70 to about 700 mg/day, for an average 70 kg human. Foradministration by perfusion fluid, a concentration of about 0.001 toabout 5 g per liter is used, preferably about 0.005 to about 2.5 g perliter, and most preferably about 0.005 to about 0.1 g per liter;perfusion can continue from tissue removal from the donor until its usefor transplantation.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

EXAMPLE 1 Protection Against Cardiac Ischaemia

This is an adaptation of the test described by Alps, et al., (Arzneim.Forsch Drug Res., 33, (1), 6, 868-876, 1983).

Eight male baboons were anaesthetized then randomly allocated to one ofthe two following groups:

Group A (control group)

Four animals were subjected to 30 min. occlusion of the left anteriordescending coronary artery (LAD) followed by a reperfusion period of 5.5hours. Venous plasma samples taken pre-thoracotomy, pre-LAD ligation andevery hour during the reperfusion period were analyzed for CPK₂ and LDH₁iso-enzyme levels.

Group B (treated group)

As in group A, except that the animals received a loading dose ofranolazine (500 ug/kg) intravenously 10 min. before LAD ligationfollowed by a continuous infusion of 50 ug/kg/min. for a 6-hour periodstarting at LAD ligation time.

RESULTS

CPK₂ iso-enzyme levels in plasma remained below the detection limitsuntil the first hour post-infarction. LDH₁ plasma levels were identicalat pre-surgery and pre-ligation times (pre-infarct period). Results, asreported in Table 1, are expressed in international units of iso-enzymeper liter of plasma.

                  TABLE 1                                                         ______________________________________                                                         Pre-       1 hr Post-                                                                             6 hr Post-                               Group   Time     Pre-infarct                                                                              reperfusion                                                                            reperfusion                              ______________________________________                                        Control CPK.sub.2                                                                              N.D.       10.5     232.8                                    group   LDH.sub.1                                                                              52.7       --       333.8                                    Tested  CPK.sub.2                                                                              N.D.       11.0     28.5                                     group   LDH.sub.1                                                                              53.0       --       85.8                                     ______________________________________                                    

As shown above, ranolazine strongly inhibited the release of CPK₂ andLDH₁, such a result being indicative of an effective protection of themyocardial tissue against deleterious effects of ischaemia.

EXAMPLE 2 Skeletal Muscle Protection

Skeletal muscle protection was determined according to the experimentalconditions as per Example 1, except that plasma samples were assayed forCPK₃ and LDH₅ iso-enzymes. The results are reported in Table 2.

                  TABLE 2                                                         ______________________________________                                                                      Post-reperfusion                                Group     Time      Pre-surgery                                                                             6 hr Post-infarct                               ______________________________________                                        Control   CPK.sub.3 104.6     2016.5                                          group     LDH.sub.5 30.0      212.0                                           Tested    CPK.sub.3 84.0      141.0                                           group     LDH.sub.5 27.4      22.3                                            ______________________________________                                    

Iso-enzyme levels are given in International Units per liter of plasma.

Thus, ranolazine clearly protected the muscle from surgery-induceddamage.

EXAMPLE 3 Myocardial Protection During Cardioplegia

This method has been described by Ferrandon et al., Br. J. Pharmacol.,93, 247P, 1988.

Male Sprague-Dawley rats were anaesthetized with pentobarbitone sodium(50 mg/kg, i.p.). After injection of heparin (200 units i.v.) the thoraxwas opened, the heart removed with a length of aorta attached and thenimmersed in ice cold Krebs' solution (118 mM NaCl, 4.55 mM KCl, 1.2 mMKH₂ SO₄, 1.2 mM MgSO₄, 11.0 mM glucose, 20.0 mM NaHCO₃, 1.35 mM CaCl₂,pH 7.4). The heart was gently palpated to expel the blood. Hearts werethen perfused with the above solution warmed to 37° C. and gassed with95% O₂ and 5% CO₂ retrogradely via the aorta (Langendorff model) using aperistaltic pump set to deliver 14 ml/min. A microelectrode wasintroduced into the ventricular muscle wall and a reference electrodeplaced in contact with the perfusion fluid 3 cm above the heart. The twoelectrodes were connected to a pH meter.

Hearts were perfused at 14 ml/min for a 15 min period to obtain a stablebaseline ventricular pH. The aortic flow was then reduced to 1 ml/minfor 15 min by decreasing the pump speed. The flow was then restored tothe initial rate for 15 min. Values of coronary flow and ventricular pHwere measured at 5 minute intervals. After restoration of the initialflow rate measurements were made at 30 seconds, 1 minute and 5 min.Samples of coronary effluent were collected and stored on ice. Infusionsof ranolazine (1 μM) were started 10 min prior to reducing the flow rateand were continued for the remainder of the experiment. At the end ofthe experiment the atria were removed and the hearts dried at 75° C. for2 days.

Biochemical determination of lactate released into the coronary effluentwas made using a spectrophotometric method. The quantity of lactatecontained in the samples was obtained by reference to a standard curve.Lactate release from the heart mass was calculated using the followingformula: ##EQU1## The results are reported in Tables 3 and 4.

                  TABLE 3                                                         ______________________________________                                        pH Modifications                                                                                  After 10 min                                                         Pre-     of         Fall in                                                   ischeamia                                                                              low perfusion                                                                            pH                                             ______________________________________                                        CONTROLS     7.36       6.77       0.59                                       RANOLAZINE   7.38       7.10       0.28                                       ______________________________________                                    

Thus, ranolazine inhibits the ischaemia-induced fall in pH byaproximately 50%.

                  TABLE 4                                                         ______________________________________                                        Lactate release modifications*                                                           2 min     5 min     15 min  1 min                                             before    after     after   after                                  Group      low perf. low perf. low perf.                                                                             reperf.                                ______________________________________                                        CONTROLS   0.6       3.34      5.0     17.4                                   1 μM    1.2       2.45      2.5     8.0                                    RANOLAZINE                                                                    ______________________________________                                         *Values are expressed as micromoles of lactate released per minute in the     coronary effluent by 1 g of dried heart.                                 

Thus, the compounds belonging to this invention clearly reduced thesequelae of low flow perfusion.

EXAMPLE 4 Protection For Organ Transplants

Twenty left nephrectomised pigs were autotransplanted with their kidneysafter preservation for 24 hours in phosphate buffered sucrose (PBS 140)and immediate contralateral nephrectomy followed theautotransplantation.

The quality of the preservation and post-transplant renal function wereassessed by measurement of glomerular filtration rate (GFR) using inulinclearance on day 7.

Group A (n=10) placebo group

The animals received placebo pre-treatment (bolus and infusion)commencing 5 min prior to left nephrectomy and lasting until the kidneywas removed. The kidney was then flushed with PBS 140 containing placebobefore storage in PBS 140. After 24 hours storage the kidney wasauto-transplanted.

Group B (n=10) treated group

The animals received a bolus dose of ranolazine intravenously (0.85mg/kg) 5 min prior to nephrectomy followed by an infusion (0.25 mg/kg/h)until the kidney was removed. The kidney was then flushed with PBS 140solution containing ranolazine 0.5 mg/l (made up immediately beforeflush) prior to storage. After 24 hours storage the kidney wasauto-transplanted.

                  TABLE 5                                                         ______________________________________                                                       Group A   Group B                                              ______________________________________                                        Glomerular filtration                                                                          16.4 ml/min 56.6 ml/min                                      rate at day 7                                                                 Peak Serum Urea  43.4 mM/1   28.5 mM/1                                        Peak Serum Creatinine                                                                          1063 μM/1                                                                              750 μM/1                                      ______________________________________                                    

The results shown in Table 5 demonstrate that organs preserved in afluid containing ranolazine achieved superior functionality aftertransplantation as compared with the control group that did not receiveranolazine.

EXAMPLE 5 Protection Against Brain Ischaemia

Isoenzyme appearance in peripheral venous blood was determined accordingto the experimental conditions as per Example 1, except that plasmasamples were assayed for CPK₁. The results are reported in Table 6.

                  TABLE 6                                                         ______________________________________                                                        CPK.sub.1 Levels                                                              Pre-surgery                                                                           6 hr post-infarct                                     ______________________________________                                        CONTROL GROUP     18.8      85.7                                              RANOLAZINE GROUP  19.9      19.3                                              ______________________________________                                    

Results are expressed in International Units per liter of plasma, andclearly demonstrate the protective role of ranolazine in cerebralischaemia.

EXAMPLE 6 Protection Against Myocardial Ischaemia

Male Sprague-Dawley rats were pentobarbitone-anaethetized andmechanically respired with room air. A left lateral thoracotomy was thenperformed and the left anterior descending coronary artery (LAD) wasoccluded for a period of 30 min. Control animals had the ligature placedin position but not tied.

Compounds (500 ug/kg ranolazine, saline vehicle) were administeredeither i.p. or i.v. 15 min prior to LAD ligation or i.p. for 3 days(twice a day) plus 15 min prior to occlusion.

At the end of the ischaemic period the ischaemic zone of the leftventricle was excised and analyzed for alpha-1-adrenoceptor densityaccording to the method described by Williams et al. (CardiovascularPharmacology, 3, 522, 1981). The apparent alpha-1-adrenoceptor densitywas calculated at 0.1 nM [³ H]-prazosin and results were expressed asfemtomole of receptors per mg of protein, as shown in Table 7. Theseresults demonstrate that ranolazine inhibits the ischaemia-inducedincrease in α-1 adrenoreceptor density in rats left ventricle and istherefore useful to prevent tissue damage resulting from myocardialischaemia.

                  TABLE 7                                                         ______________________________________                                                                            Alpha-1                                   Group         Route     No. Animals density                                   ______________________________________                                        Control                 12          8.65                                      Ischaemia/Treated by    12          16.30                                     saline vehicle only                                                           Ischaemia/Treated by                                                                        i.p.      13          11.20                                     ranolazine                                                                    Ischaemia/Treated by                                                                        i.v.      9           9.71                                      ranolazine                                                                    Ischaemia/Treated by                                                                        i.p. 3 days                                                                             9           8.33                                      ranolazine                                                                    ______________________________________                                    

EXAMPLE 7 Formulations

The following example illustrates the preparation of representativepharmaceutical formulations containing a compound of Formula I, asexemplified by ranolazine.

    ______________________________________                                        A. I.V. FORMULATION (low concentration)                                       (ranolazine)       5.0 mg      0.5 g                                          dextrose monohydrate                                                                             51.2 mg     5.1 g                                          sodium hydroxide q.s. to                                                                         pH 4        pH 4                                           water for injection to                                                                           1.0 ml      100 ml                                         B. I.V. FORMULATION (high concentration)                                      (ranolazine)       200.0 mg    2 g                                            dextrose monohydrate                                                                             39.4 mg     4 g                                            sodium hydroxide q.s. to                                                                         pH 4        pH 4                                           water for injection to                                                                           1.0 ml      100 ml                                         ______________________________________                                    

To prepare the I.V. formulations, ranolazine and dextrose monohydrateare dissolved into water (70 per cent of the final desired volume) thensodium hydroxide (10N solution) is added under stirring until pH 4 andthe volume is completed to 100 ml with water. The medium is filteredthrough a 0.2 micron membrane filter and packaged in ampoules or vialsunder sterile conditions. Alternatively the medium can be filtered undernon-sterile conditions, packed in ampoules then sterilized byautoclaving.

    ______________________________________                                        C. FILM COATED TABLET FORMULATION                                             Ingredients        Parts by weight                                            ______________________________________                                        ranolazine HCl (A) 80.0                                                       microcrystalline cellulose (B)                                                                   16.5                                                       polyvinylpyrrolidone (C)                                                                         1.0                                                        crosscarmellose sodium (D)                                                                       2.0                                                        magnesium stearate (E)                                                                           0.5                                                        ______________________________________                                    

(A), (B) and half of (D) are mixed then (C) and water are added to allowwet granulation. (E) and the remaining part of (D) are finally added.After careful mix the granulated mixture is dried, formed into tabletscontaining up to 250 mg of active compound, and the tablets are filmcoated using White Opadry following appropriate techniques.

    ______________________________________                                        D. CONTROLLED RELEASE FORMULATION                                             Ingredients        Parts by weight                                            ______________________________________                                        ranolazine BASE (A)                                                                              90                                                         microcrystalline cellulose (B)                                                                   10                                                         ______________________________________                                    

The two above ingredients are dry mixed then water is added to form awet mass adequate for extrusion then spheronisation (0.5 to 1.4 mm).Microspheres are coated with appropriate release-controlling polymersthen put into hard shell capsules containing up to 250 mg of activeingredient per unit.

    ______________________________________                                        E. PERFUSION FLUID                                                            Ingredients            Parts by weight                                        ______________________________________                                        Ranolazine             20        mg                                           Phosphate Buffered Sucrose:                                                   Sucrose                48.0      g                                            Sodium Dihydrogen Phosphate                                                                          4.59      g                                            Sodium Monohydrogen Phosphate                                                                        6.53      g                                            Water For Injection (U.S.P.)                                                                         q.s. to 1000                                                                            ml                                           ______________________________________                                    

The ingredients are dissolved in a portion of the Water For Injection,and once dissolved, the remaining volume is made up with Water ForInjection.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method of treating tissues experiencing aphysical or chemical insult, said method comprising: administering,toprotect skeletal muscles against damage resulting from trauma orsubsequent to muscle or systemic diseases, to treat shock conditions, orto preserve donor tissues used in transplants, a therapeuticallyeffective amount of a compound of the formula: ##STR2## or apharmaceutically acceptable ester or acid addition salt thereof,wherein: R¹, R², R³, R⁴ and R⁵ are each independently hydrogen, loweralkyl, lower alkoxy, cyano, trifluoromethyl, halo, lower alkylthio,lower alkyl sulfinyl, lower alkyl sulfonyl, N-optionally substitutedalkylamido, except that when R¹ is methyl, R⁴ is not methyl; or R² andR³ taken together form --OCH₂ O--; R⁶, R⁷, R⁸, R⁹ and R¹⁰ are eachindependently hydrogen, lower acyl, aminocarbonylmethyl, cyano, loweralkyl, lower alkoxy, trifluoromethyl, halo, lower alkylthio, lower alkylsulfinyl, lower alkyl sulfonyl, di-lower alkyl amino; R⁶ and R⁷ togetherform --CH═CH--CH═CH--; or R⁷ and R⁸ together form --OCH₂ O--; R¹¹ andR¹² are each independently hydrogen or lower alkyl; and W is oxygen orsulfur.
 2. The method of claim 1 wherein R¹ and R⁵ are methyl.
 3. Themethod of claim 2 wherein R², R³, R⁴, R¹¹ and R¹² are hydrogen.
 4. Themethod of claim 3 wherein W is oxygen.
 5. The method of claim 4 whereinR⁶ is methoxy and R⁷, R⁸, R⁹ and R¹⁰ are hydrogen.
 6. The method ofclaim 1 comprising a method for treating shock conditions, includingcardiogenic shock.
 7. The method of claim 6 wherein said compound isranolazine, or a pharmaceutically acceptable salt thereof.
 8. The methodof claim 1 comprising a method for preserving donor tissues used intransplants, by administration to the donor, the recipient and/or byadding to the ex-vivo perfusion fluid an effective amount of saidcompound.
 9. The method of claim 8 comprising a method for preservingdonor tissues used in renal transplants, skin grafts, cardiactransplants, lung transplants, corneal transplants, or livertransplants.
 10. The method of claim 8 wherein said compound isranolazine, or a pharmaceutically acceptable salt thereof.
 11. Themethod of claim 9 wherein said compound is ranolazine, or apharmaceutically acceptable salt thereof.
 12. A perfusion solution forpreserving donor tissue for transplantation comprising a therapeuticallyeffective amount, up to 5 mg/ml, of a compound of the formula: ##STR3##or a pharmaceutically acceptable ester or acid addition salt thereof,wherein:R¹, R², R³, R⁴ and R⁵ are each independently hydrogen, loweralkyl, lower alkoxy, cyano, trifluoromethyl, halo, lower alkylthio,lower alkyl sulfinyl, lower alkyl sulfonyl, N-optionally substitutedalkylamido, except that when R¹ is methyl, R⁴ is not methyl; or R² andR³ together form --OCH₂ O--; R⁶, R⁷, R⁸, R⁹ and R¹⁰ are eachindependently hydrogen, lower acyl, aminocarbonylmethyl, cyano, loweralkyl, lower alkoxy, trifluoromethyl, halo, lower alkylthio, lower alkylsulfinyl, lower alkyl sulfonyl, di-lower alkyl amino; R⁶ and R⁷ togetherform --CH═CH--CH═CH--; or R⁷ and R⁸ together form --OCH₂ O--; R¹¹ andR¹² are each independently hydrogen or lower alkyl; and W is oxygen orsulfur,in a donor tissue perfusion solution carrier.
 13. The perfusionsolution of claim 12 wherein said compound is ranolazine, or apharmaceutically acceptable salt thereof.
 14. The perfusion solution ofclaim 13 comprising from 0.0001 g to 5 g of ranolazine, or apharmaceutically acceptable salt thereof, in 1000 ml of phosphatebuffered sucrose.
 15. A method of treating hypoxic injury to skeletalmuscle, said method comprising: administering to a mammal in needthereof a therapeutically effective amount of a compound of the formula:##STR4## or a pharmaceutically acceptable ester or acid addition saltthereof, wherein:R¹, R², R³, R⁴ and R⁵ are each independently hydrogen,lower alkyl, lower alkoxy, cyano, trifluoromethyl, halo, loweralkylthio, lower alkyl sulfinyl, lower alkyl sulfonyl, N-optionallysubstituted alkylamido, except that when R¹ is methyl, R⁴ is not methyl;or R² and R³ taken together form --OCH₂ O--; R⁶, R⁷, R⁸, R⁹ and R¹⁰ areeach independently hydrogen, lower acyl, aminocarbonylmethyl, cyano,lower alkyl, lower alkoxy, trifluoromethyl, halo, lower alkylthio, loweralkyl sulfinyl, lower alkyl sulfonyl, di-lower alkyl amino; R⁶ and R⁷together form --CH═CH--CH═CH--; or R⁷ and R⁸ together form --OCH₂ O--;R¹¹ and R¹² are each independently hydrogen or lower alkyl; and W isoxygen or sulfur.
 16. The method of claim 15 wherein said compound isranolazine or a pharmaceutically acceptable salt thereof.
 17. The methodof claim 16 comprising administering ranolazine or a pharmaceuticallyacceptable salt thereof in an amount effective for preventing,inhibiting or relieving hypoxic reperfusion injury to skeletal musclescaused by peripheral vascular disease.
 18. The perfusion solution ofclaim 12 wherein the donor tissue perfusion solution carrier is selectedfrom Eurocollins Solution, University of Wisconsin Fluid, phosphatebuffered sucrose, and Hyperosmolar Citrate.
 19. The perfusion solutionof claim 18 wherein said compound is ranolazine, or a pharmaceuticallyacceptable salt thereof.